Hepatocyte growth factor activator (HGFA) is a plasma trypsin-like serine protease secreted mainly by the liver that regulates the mitogenic, motogenic, and morphogenic activities of hepatocyte growth factor (HGF, also known as scatter factor (SF)) (Shimomura et al., Cytotech., 8:219-229 (1992)). HGF is implicated in embryonic development, tissue regeneration and invasive tumor growth. This activity requires proteolytic processing of HGF into a two-chain, disulfide-linked α,β-heterodimeric form. HGFA is among the most potent activators of HGF identified so far. (Shimomura et al., Eur. J. Biochem. 229 (1995)). HGFA expression has been reported in normal gastrointestinal renal tissues, and in the central nervous system, as well as in pancreatic, hepatocellular, colorectal, prostatic, and lung cancer cells. (Itoh et al., Biochim. Biophys. Acta, 1491:295-302 (2000); van Adelsberg et al., J. Biol. Chem., 276:15099-15106 (2001); Hayashi et al., Brain Res., 799:311-316 (1998); Moriyama et al., FEBS Lett., 372:78-82 (1995); Parr et al., Int. J. Oncol., 19:857-863 (2001); Kataoka et al., Cancer Res., 60:6148-6159 (2000); Nagata et al., Biochem. Biophys. Res. Comm., 289:205-211 (2001)). Recently, HGFA secretion from multiple myeloma cells has been linked to the potent para- and/or autocrine effect of HGF. (Tjin et al., Blood, 104:2172-2175 (2004)).
HGFA is secreted as a 96 kDa zymogen (proHGFA) with a domain structure like that of coagulation factor XII (FXIIa), comprising 6 domains. Those domains include an N-terminal fibronectin type II domain, an epidermal growth factor (EGF)-like domain, a fibronectin type 1 domain, another EGF-like domain, a kringle domain, and a C-terminal trypsin homology serine protease domain. (Miyazawa, et al., J. Biol. Chem., 268:10024-10028 (1993)). Cleavage at a kallikrein-sensitive site between residues Arg372 and Va1373 can produce a short 34 kDa form that lacks the first 5 domains. Both the 96 kDa and 34 kDa forms of proHGFA can be cleaved between residues Arg407 and Ile408 into active HGFA by thrombin. (Shimomura et al., J. Biol. Chem., 268, 22927-22932 (1993)). Thrombin is the ultimate effector of pro-coagulant stimuli and generation of active HGFA would be consistent with the activity of HGF in wound repair. (Bussolino et al., J. Cell Biol., 119:625-641 (1992)).
Among factors influencing HGF/Met signaling are the activation of proHGFA and subsequent inhibition of HGFA. The identified physiological inhibitors of HGFA are the splice variants HAI-1 and HAI-1B (hepatocyte growth factor activator inhibitor-1), and HAI-2 (also known as placental bikunin) (Shimomura et al., J. Biol. Chem., 272:6370-6376 (1997); Kawaguchi et al., J. Biol. Chem., 272:27558-27564 (1997); Kirchhofer et al., J. Biol. Chem. 278:36341-36349 (2003)). HGFA has restricted substrate specificity (Kataoka et al., Cancer metastasis reviews 22, 223-239 (2005); Miyazawa et al., J Biol Chem 268, 10024-10028 (1993)): only two macromolecular substrates, pro-hepatocyte growth factor (pro-HGF) (Shinomura et al., Eur J Biochem 229, 257-261 (1995) and pro-macrophage stimulating protein (pro-MSP) (Kawaguchi et al, Febs J 276(13)3481-3490 (2009), are known to be processed by HGFA, exemplifying the enzyme's highly restricted substrate specificity. HGFA is inhibited by the Kunitz-type inhibitor HGFA inhibitor-1, which utilizes the N-terminal Kunitz domain-1 (KD1) to inhibit HGFA by a canonical inhibition mechanism (Shia et al., J Mol Bio 346, 1335-13492005). HGFA effects tissue regeneration and promotes cancer growth via pro-HGF processing and ensuing activation of the HGF/Met signaling pathway (Parr and Jiang, Int'l J of Oncol 19, 857-863 (2001)).
Allosteric regulation of an enzyme, by definition, involves an altered catalytic activity originating from a remote effector interaction site. In fact, all dynamic proteins (monomeric and multimeric) seem to have a potential for allosterism (Gunasekaran et al., Proteins 57, 433-443 (2004)). Elucidation of allosteric modulation and its pathways of communication have received considerable attention (Swain and Gierasch, Curr Op in Structural Biol 16, 102-108 (2006); Yu and Koshland, PNAS 98, 9517-9520 (2001)). A classic example of allostery is observed in hemoglobin (Perutz, Nature 228, 726-739 (1970)), which offered the first mechanistic insights on allosteric regulation. Several X-ray crystallographic studies emerged thereafter describing the conformational changes during allosteric regulation (Changeux and Edelstein, Science 308, 1424-1428 (2005); Di Cera, J Biol Chem 281, 1305-1308 (2006); Pellicena and Kuriyan, Nature 228, 726-739 (2006); Xu et al., Nature 388, 741-750 (1997)). Allostery is also a quite common and powerful mechanism to regulate the catalytic activity of proteases (Hauske et al., Chembiochem 9, 2920-2928 (2008); Turk, Nature Reviews 5, 785-799 (2006)). Unlike active sites, distally located allosteric sites are usually less conserved and can be exploited to achieve specificity (Hauske et al., supra). Allosteric anti-protease protein-based agents have great therapeutic potential, since they are potent and highly specific and are safeguarded from any inadvertent processing by their target protease. Examples of allosteric regulators in the serine protease family (Clan PA, Family S1 in MEROPS nomenclature (Rawlings et al., Nucleic Acids Res 36, D320-325 (2008))) are the accessory PDZ domains in the HtrA protease family (Sohn et al., Cell 131, 572-583 (2007)), calcium for many coagulation factors (Bjelke et al., J Biol Chem 283, 25863-25870 (2008)), sodium for thrombin (Huntington, Biological chemistry 389, 1025-1035 (2008); Wells and Di Cera, Biochem 31, 11721-11730 (1992)), cofactors such as tissue factor for coagulation factor VIIa (Eigenbrot and Kirchhofer, Trends in Cardiovascular Med 12, 19-26 (2002)) and N-terminal peptide insertion into the “activation pocket” (Friedrich et al., Nature 425, 535-539 (2003); Huber and Bode, Acc Chem Res 11, 114-122 (1978)).
Proteases have been implicated in many human pathological processes (Barrett et al., (1998). Handbook of Proteolytic Enzymes. San Diego: Academic Press (1998); Egeblad and Werb, Nature Rev Cancer 2, 161-174 (2002); Hooper, Proteases in Biology and Medicine. In Essays in Biochemistry, London: Portland Press (2002); Luttun et al., Curr Atheroscler. Rep 2, 407-416 (2000)). Therefore, regulation of proteolytic activity by allosteric inhibitors might represent a promising alternative approach to active site inhibitors (Peterson and Golemis, J Cell Biochem 93, 68-73 (2004)), which often suffer from inadequate specificity, since active site topologies are generally conserved among members of the same family (Hedstrom, Chem Revs 102, 4501-4524 (2002)). Unlike active sites, distally located allosteric sites are usually less conserved and can be exploited to achieve specificity (Hauske et al., supra). Excellent examples of specific and potent allosteric inhibitors have been described for coagulation factor VIIa and caspases (Hardy et al., PNAS 101, 12461-12466 (2004); Hardy and Wells, Curr Op Structural Biol 14, 706-715 (2009)).
Since activation of pro-HGF requires cleavage by a convertase such as HGFA, modulation of HGFA function and/or its interaction with its substrate could prove to be an efficacious therapeutic approach. In this regard, there is a clear need to identify clinically relevant agents capable of modulating activity of and/or specifically interacting with HGFA. The invention fulfills this need and provides other benefits.
All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference.